Method of promoting bone growth with hyaluronic acid and growth factors

ABSTRACT

A bone growth-promoting composition is provided comprising hyaluronic acid and a growth factor. The composition has a viscosity and biodegradability sufficient to persist at an intra-articular site of desired bone growth for a period of time sufficient to promote the bone growth. Preferably hyaluronic acid is used in a composition range of 0.1-4% by weight and preferred growth factor is bFGF, present in a concentration range of about 10 −6  to 100 mg/ml.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. Ser. No. 09/360,543, filed Jul. 26, 1999, nowU.S. Pat. No. 6,221,854, which is a continuation-in-part of U.S. Ser.No. 08/811,971, filed Mar. 5, 1997, now U.S. Pat. No. 5,942,499 which isa continuation-in-part of Ser. No. 08/611,690, filed Mar. 5, 1996, nowabandoned all incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a naturally-occurring polysaccharide containingalternating N-acetyl-D-glucosamine and D-glucuronic acid monosaccharideunits linked with beta 1-4 bonds and the disaccharide units linked withbeta 1-3 glycoside bonds. It occurs usually as the sodium salt and has amolecular weight range of about 50,000 to 8×10⁶.

SUMMARY OF THE INVENTION

The present invention provides a bone growth-promoting compositioncomprising hyaluronic acid and a growth factor such that the compositionhas a viscosity and biodegradability sufficient to persist at the siteof desired bone growth for a period of time sufficient to promote bonegrowth.

Compositions comprising hyaluronic acid and a growth factor are providedwhich have the requisite viscosity and biodegradability.

As used herein, the term hyaluronic acid, abbreviated as HA, meanshyaluronic acid and its salts such as the sodium, potassium, magnesium,calcium, and the like, salts.

By growth factors, it is meant those factors, proteinaceous orotherwise, which are found to play a role in the induction or conductionof growth of bone, ligaments, cartilage or other tissues associated withbone or joints.

In particular these growth factors include bFGF, aFGF, EGF (epidermalgrowth factor), PDGF (platelet-derived growth factor), IGF (insulin-likegrowth factor), TGF-β I through III, including the TGF-β superfamily(BMP-1 through 12, GDF 1 through 12, dpp, 60A, BIP, OF).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of experimental data set forth inexample 1 below; FIG. 1A shows the bone thickness formed as a functionof bFGF dosage; FIG. 1B shows bone thickness formation as a function ofhyaluronic acid concentration;

FIG. 2 is a graphical representation of the experimental data set forthin Example 2 below.

FIG. 3 is a graphical representation of the load at failure of healingrabbit fibula after 23 and 30 days following treatment according toExample 3.

FIG. 4 is a graphical representation of the energy to failure (inpounds) of healing rabbit fibula after 23 and 30 days followingtreatment according to Example 3.

FIG. 5 is a graphical representation of the bone thickness data in ratsfollowing treatment according to Example 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The processes by which the compositions and the method of their use aredescribed in more detail.

The HA is preferably uncrosslinked having a molecular weight of 500,000and above, typically in the range of 10⁴ to 10⁷. The bonegrowth-promoting compositions will typically contain from about 0.1 upto 4 percent by weight of uncrosslinked HA in an aqueous solution whichalso contains other solution excipients such as buffer salts, sugars,anti-oxidants and preservatives to maintain the bio-activity of thegrowth factor and proper pH of the composition. A composition containingfrom about 0.1 to 2 percent by weight of uncrosslinked HA is preferred.A typical pH of the solution will be in the range of 4 to 9, preferablyabout 6.0±1.0 and most preferably about 5.0.

The growth factor will typically be present in the solution in aconcentration range of about 10⁻⁶ to 100 mg/ml of solution, particularlyin the case of bFGF preferably about 0.1 to 20 mg/ml. The concentrationwill be dependent upon the particular bone site and application, as wellas the volume of the injection and specific activity of the growthfactor. An intra-articular site is preferred.

It is important for the solution used to promote the growth to have aviscosity which allows it to be injectable through a syringe orcatheter, but not to be prematurely diluted by the body fluids beforethe bone promoting effect can be achieved. Preferably, the viscosity ofthe composition is within a range of 10 to 10⁶ cP and, in the case ofbFGF-containing compositions, preferably about 75,000 cP.

It is also important for the composition to have a biodegradabilitywhich is sufficient to allow it to remain in place at the site ofdesired bone growth to effect the bone growth-promoting activity.

The composition must usually persist at the site of desired bone growthfor a period from about three (3) to about thirty (30) days, typicallyfrom three (3) to about fourteen (14) days. If the composition isdispersed prematurely, the desired bone growth-promotion effect eitherwill not occur or the formed bone will not have the desired strength.

If the composition persists at the site of desired bone growth for anexcessive period, its presence at the bone site may inhibit the naturaldevelopment of the bone, sometimes resulting in no bone formation atall.

The compositions are typically formed as solutions by mixing the HA andgrowth factor in appropriate amounts of excipients such as sodiumcitrate, EDTA and sucrose so that the HA and growth factor remain insolution at the desired concentration and the solution exhibits theappropriate viscosity and biodegradability. The solution may be appliedto the site of desired bone growth in any convenient manner, typicallyby introduction through a syringe or catheter. Administration at anintra-articular site is preferred, where there is a bone joint.

Administration of a bone growth composition of the present invention maybe desirable to accelerate wound healing, prevent further tissue damageoccurring subsequent to injury, avoid treatments that compromise thenatural healing process and create optimal physical and biologicalconditions for healing. Sites of desired bone growth includetibia/fibula fractures; femur/humerus fractures; forearm fractures;posteriorly displaced distal radius (Colles) fracture; stress fracturesincluding sports fractures associated with shin splints and footinjuries; vertebral compression fractures, rib fractures and clavicularfractures. Sites of desired bone growth also include pathological bonedefects associated with osteoporosis, osteomalacia, hyperparathyroidism,renal osteodystrophy, and primary and metastatic cancer of the bone.

The invention is described in more detail in the following examples,which are provided by way of illustration and are not intended to limitthe invention set forth in the claims.

EXAMPLE 1

Sodium hyaluronate (Genzyme, MW 2×10⁶, sterile, viscosity in 1% solutionof 6500 cP), bFGF (Scios-Nova, 4.3 mg/ml solution (pH 5) in 9% sucrose,20 mM sodium citrate and 1 mM EDTA) were mixed. The formulations wereformed by mixing sterile-filtered solutions of bFGF and other excipients(sodium citrate, water, etc.) with the appropriate amount of solid,sterile HA. The HA was dispersed quickly by repeated back and forthsyringing to prevent the formation of large aggregates of particles.Formulations were prepared aseptically and administered in prefilled 1ml plastic syringes with 21 G needles into male Sprague-Dawley rats (8-9weeks old, 160-180 grams), which were anesthetized with acepromazine,xylazine and ketamine. A 5-10 millimeter incision was made laterally inthe skin at the back of the neck to locate the intersection of thesagittal and lambdoid sutures. Fifty microliters of the test formulationwas injected with a 21 G needle between the periosteum and parietalbone. The animals were euthanized 14 days following treatment.

Tissues for histological analysis were fixed in 10% neutral bufferedformalin. Tissues were decalcified for at least 2 hours in formic acid(RapidBone Decal) with constant, gentle agitation. Samples weredehydrated and infiltrated with paraffin. Specimens were then embeddedin a cross-sectional plane and sectioned at 5 μm. Sections were stainedwith hematoxylin and eosin for histological analysis. New bone formationwas scored on a scale of 0 to 4 as shown in Table 1.

TABLE 1 Qualitative description of new, woven bone formation on parietalbone following subperiostal injection. Score Description of New, WovenBone Formation 0 None; no new, woven bone 1 Trace/patchy areas of wovenbone 2 Areas of both continuous and patchy woven bone 3 Thin, continuouswoven bone (<50% of original parietal bone) 4 Thick, continuous wovenbone (>50% of original parietal bone)

The total thickness of the parietal bone was measured similar to themethod of Noda et al., Endocrinology, 124:2991-4, 1989. A photograph ofeach histology section was taken 2 to 3 mm lateral to the sagittalsuture (the approximate midpoint between the sagittal suture and theedge of the section). Three bone thickness measurements of total bonewere taken at the left, middle, and right side of the photograph andscaled to determine total bone thickness. Both dense cortical bone andnew, woven bone were included in the measurement.

In all groups the response to each treatment was consistent betweenanimals in the same treatment group. Qualitatively, the groups ofanimals treated with all of the bFGF/HA gel formulations exhibited newbone formation while placebo treated and growth control animals showminimal or no new bone formation (table 2). It was apparent that onlysmall differences existed between the bFGF/HA formulations examined inthis study. However, there did appear to be a dose response effect.(FIGS. 1A, 1B).

TABLE 2 Qualitative results of histological scoring (table 1) of animalsreceiving subperiosteal injections of bFGF formulations 14 daysfollowing treatment. Formulation Total number of animals bFGF Dose HAConc. with a bone formation score of: (μg) (%) 0 1 2 3 4 100 2   2 2 — —— — 100 2   2 2 — — — —  10 2   2 2 100 0.5 1 2 1 100 0.1 2 — 2   2 sham3 operation growth 2 control

Table 3 shows the total bone thickness of the rat calvaria afterreceiving different formulations by subperiosteal injection. Allformulations containing bFGF and HA exhibited new bone formation. Thefirst two entries in table 3 represent replicate experiments. Replicategroups of animals receiving 100 μg bFGF in a 2% HA gel had a totalparietal bone thickness of 0.49±0.10 mm in the first study and 0.59±0.12mm in the second study, a 17% difference. However, the total bonethickness of both groups was qualitatively and quantitativelysignificantly different than control. All formulations containing 100 μgof bFGF and HA had at least a 61% increase in new bone formationcompared to animals receiving no treatment.

FIGS. 1A and 1B show the effect of bFGF and HA concentration on totalbone thickness. As the dose of bFGF increase from 10 to 100 μg, thetotal bone thickness increases 20% from 0.45 to 0.54 mm. As theconcentration of HA increases, an increase in total bone formation isseen until a maximum increase in bone formation is observed near 0.5%HA; increasing the concentration of HA above 0.5% does not result in anadditional increase in new bone formation elicited by bFGF in this model(FIG. 1B).

TABLE 3 The total bone thickness of a section of the rat calvaria 2 mmanterior of the lambda and 2 to 3 mm lateral to the parietal suture 14days following treatment. Bone thickness is the average of 3measurements per animal. n is the number of replicate animals, and thepercent increase represents the fractional increase over growth control.Formulation Total Bone Thickness bFGF Dose HA Conc. mean ± SD Percent(μg) (%) n (mm) Increase 100 2   4 0.49 ± 0.10  75% 100 2   4 0.59 ±0.12 111% — —  10 2   4 0.45 ± 0.07  61% — — 100 0.5 4 0.55 ± 0.09  96%100 0.1 4 0.46 ± 0.16  64% 100 — 2 0.34 ± 0.04  21% — 2   4 0.33 ± 0.04 18% sham 3 0.24 ± 0.04 −14% operation growth 2 0.28 ± 0.01  0% control

It was thus shown that a single, subperiosteal injection of 100 μg ofbFGF in an HA gel showed significant qualitative and quantitative effecton intramembranous bone formation over controls. Fourteen days followingadministration, up to 111% new bone is formed at the site of injectionin animals treated with 100 μg of bFGF in HA gels. Placebo and controlgroups all had less than a 18% increase in bone thickness 14 daysfollowing injection. As the dose of bFGF increase from 10 to 100 μg, thetotal bone thickness increases 20% from 0.45 to 0.54 mm. Increasing theconcentration of HA above 0.5% does not result in an additional increasein new bone formation elicited by bFGF in this model.

EXAMPLE 2

The tests described in Example 1 were conducted using 8 differentformulations. The bFGF was used in combination with hyaluronic acid ascompared to 7 other compositions wherein bFGF was used with othercarriers or the carriers were used alone as placebos. The results areshown below and are summarized in FIG. 2 and Table 4.

TABLE 4 The total number of animals with a bone formation score Totalnumber of animals with a bone formation socre of: Formulation 0 1 2 3 4100 μg bFGF in 2% HA 3 1 2% HA placebo 4 100 μg bFGF IN 2% collagen 2 2(CSF) 2% collagen (CSF) placebo 3 1 100 μg bFGF in 2% Dex. Sulf. 1 1 22% Dex. Sulf. placebo 4 100 μg bFGF in 2% Ficoll* 3 1 2% Ficoll placebo4 *An uncharged polysaccharide.

EXAMPLE 3

Formulations of sodium hyaluronate (2%) and bFGF (4 mg/ml) were preparedas in Example 1 for administration to a fracture site in rabbits. Aformulation was also prepared containing 4 mg/ml bFGF, 6 mg/ml rabbitfibrinogen, 0.2 mg/ml aprotinin, and other excipients to maintain pH andstability. This fibrinogen formulation was similar to a previouslypublished composition used for fracture repair¹. A 1 mm cut in thefibula mid-diaphysis was surgically created in New Zealand White rabbitsto model a bone fracture. This experimental method has previously beenutilized to examine the healing of fractures in rabbits². Animals weretreated with 50 μL of the HA/bFGF formulation, 50 μL of thefibrinogen/bFGF formulation, or remained untreated.

The mechanical strength of 10 healed fibula per treatment group wasmeasured by a four point bending technique 23 days following treatment.FIG. 3 illustrates the load at failure for untreated, HA/bFGF treated,and fibrinogen/bFGF treated fibulae. The HA/bFGF treated fibulae were53% stronger than untreated control, while the fibrin/bFGF treatedfibulae were 30% stronger than untreated control. FIG. 4 shows theenergy to failure for all three treatment groups. By this measurement,the HA/bFGF treated fibulae were 43% stronger than untreated control,while the fibrin/bFGF treated fibulae were 3% weaker than untreatedcontrol.

In addition, the mechanical strength of 10 untreated fibulae and 10HA/bFGF treated fibulae was measured 30 days following treatment. FIG. 3shows that the load at failure is 36% higher in HA/bFGF treated animalsover control and that this difference is statistically significant(p=0.02). FIG. 4 indicates that the energy to failure is 79% higher inthe HA/bFGF versus control and that this difference is statisticallysignificant (p=0.01). FIGS. 3 and 4 also show that the strength ofHA/bFGF treated fibulae return to the strength of intact bone morequickly than untreated fibulae, indicating accelerated bone healing.

1. Hiroshi Kawaguchi, et al., Stimulation of Fracture Repair byRecombinant Human Basic Fibroblast Growth Factor in Normal andStreptozotocin-Diabetic Rats, Endocrinology, 135:774-781, 1994.

2. A. A. Pilla, et al., Non-invasive Low-intensity Pulsed UltrasoundAccelerates Bone Healing in the Rabbit, Journal of Orthopaedic Trauma,4:246-253, 1990.

EXAMPLE 4

The method in Example 1 was used to compare total bone formation of theHA/bFGF formulation in Example 1, the fibrin/bFGF formulation in Example3, and a bFGF in an aqueous sucrose/citrate buffer formulation. 100 μgof bFGF in 50 μL each formulation was administered by subperiostealinjection, and animals were sacrificed 7 and 14 dayspost-administration. In addition, animals receiving no treatment wereused as controls.

In each of the four groups the response to each treatment was veryconsistent between animals. At 7 days all bFGF treated animals showintramembranous bone that has formed on the calvarium in response to thebFGF. The control animals show minimal or no new bone formation.Qualitatively, the group of animals treated with bFGF in a HA gel hadmore new bone formation than in any of the other bFGF formulations. Inthe 14 day specimens, the difference in the amount of bone formation inthe bFGF/HA gel treated animals was even more apparent. While new boneformed in all bFGF treated animals, it was readily apparent that a muchthicker bone mass had formed in the bFGF/HA gel treated animals than inany other treatment group.

FIG. 5 shows the quantitative results of the bone thickness measurement.The thickness 7 days after treatment is 95% thicker in the animalsadministered 100 μg of bFGF in a 2% HA gel than in animal receiving notreatment (i.e. control). The other bFGF treated groups showed a 86 and55% increase in bone formation by treatment with bFGF in a fibrin geland bFGF in an aqueous citrate buffer, respectively.

At 14 days 111% new bone is formed in animals treated with 100 μg of FGFin an HA gel (FIG. 5). Other bFGF treated groups had only a 25 and 21%increase in bone formation in rats treated with bFGF in a fibrin gel andbFGF in an aqueous citrate buffer, respectively.

EXAMPLE 5

The effect of the molecular weight of hyaluronic acid in basicfibroblast growth factor (bFGF) formulations on intramembranous boneformation was examined by subperiosteal injection to the rat parietalbone.

Materials and Methods

The HA with a molecular weight of 760 to 2300 KDa (from Genzyme andLifecore Biomedical) was used to prepare formulations. The BFGF wasprovided (Scios-Nova) as a frozen solution (4.3 mg/ml) in 9% sucrose, 20mM sodium citrate, and 1 mM EDTA adjusted to pH 5.0. Other reagents(sucrose, sodium citrate, EDTA) were purchased from Sigma.

Formulations were prepared by mixing a sterile filtered solution of bFGF(2 mg/ml) with the appropriate amount of HA (20 mg/ml). The solution andcarrier initially were in separate syringes connected by a stopcock. Theformulation was mixed by repeated back and forth syringing. Formulationswere prepared aseptically and administered in prefilled 1 ml plasticsyringes with a 21 G needle.

Male Sprague-Dawley rats (8-9 weeks old, 160-180 g) were anesthetizedwith a mixture of acepromazine, xylazine, and ketamine. A small incision(5-10 mm) was made laterally in the skin at the back of the neck. Theintersection of the sagittal and lambdoid sutures was located, and 50 μLof each formulation was injected with 21 G needle on the left sidebetween the periosteum and parietal bone. Fourteen days followingtreatment animals were euthanized by CO₂ asphyxiation.

Tissues for histological analysis were fixed in 10% neutral bufferedformalin. Tissues were decalcified for at least 2 hours in formic acid(RapidBone Decal) with constant, gentle agitation. Samples weredehydrated and infiltrated with paraffin. Specimens were then embeddedin a cross-sectional plane and sectioned at 5 μM. Sections were stainedwith hematoxylin and eosin for histological analysis. New bone formationwas scored on a scale of 0-4. A score of 0 represented no new wovenbone; a score of 1 represented trace or patchy areas of woven bone; ascore of 2 represented larger areas of patchy bone formation; a score of3 represented thin, continuous woven bone (<50% of original parietalbone) and a score of 4 represented thick, continuous woven bone (>50% oforiginal parietal bone).

Bone Thickness Measurement

The total thickness of the parietal bone was determined at the site ofinjection. A photograph of each histology section was taken 2 to 3 mmlateral to the sagittal suture (the approximate midpoint between thesagittal suture and the edge of the section). Three bone thicknessmeasurements of total bone were taken at the left, middle, and rightside of the photograph and scaled to determine total bone thickness.Both dense cortical bone and new, woven bone were included in themeasurement.

Results

Qualitatively, all groups of animals treated with bFGF exhibited somenew bone formation while HA only gel treated animals and controls showminimal or no new bone formation. Histologically, bFGF treated animalsshowed the presence of new, woven bone and mature lamellar bone. At theinjection site a marked layer of new woven bone had formed superficialto the more mature underlying bone occasionally, the woven bone ispresent on the right side, but is not present to the same extent that isseen on the left side. The new woven bone show normal reversal lines,marrow spacing and general staining characteristics. Most animals inthese groups received a bone formation score of 3 (28/30), while twoanimals received the maximum score of 4. Above the woven bone, there isan area of adipose and fibrous tissue in close approximation to the newwoven bone and appear normally configured. No areas show foci of chronicinflammatory cells which would be an indication of antigenic potentialof the HA/bFGF formulation.

The HA gel treated animals showed no or very little new bone formation,and most animals received a bone formation score of 0 (26/30). Three of30 animals had a bone formation score of 1 while a single animal had abone formation of 3. The new bone formation may be a result of elevationof the periosteum during the surgical procedure. No abnormalities areobserved in any part of the tissue, and there is no indication ofantigenic potential in any of the HAs examined.

Animals receiving no surgery and no treatment showed no new boneformation and all six animals received a bone formation score of zero.This group was very similar to the groups receiving HA gel, except thatno new bone formed as a result of elevation of the periosteum. Thespecimens consisted of mature bone in which normal osteocytes arepresent in lacunae, and marrow spaces were seen. Small amounts of finefibrous tissue are present superficially to the bone tissue in allsections.

With respect to bone thickness, FGF treated groups had a 68-100%increase in bone thickness over the growth control. The animals treatedwith bFGF in a gel formed from Lifecore's highest molecular weight HAavailable had the largest increase in bone thickness (100%). There was aslight effect of molecular weight on bone formation. As the molecularweight of HA increased, the amount of new bone formed also increased.This increase in bone formation could be due to the increase inviscosity of the formulation. As the viscosity increased, it became alarger diffusional barrier for the FGF maintaining it at the sitelocally for a longer period. The longer residence time of HA thenresults in more bone formation.

EXAMPLE 6

This Example addresses the local distribution and persistence ofhyaluronic acid following subperiosteal injection of an HA+bFGF gel.This study examined the proliferation of the periosteum, new boneformation, and the local distribution and persistence of hyaluronic acid(HA) following subperiosteal injection of an HA gel containing basicfibroblast growth factor (bFGF). The periosteal thickness at 3 days andbone thickness at 10 days was determined by histologic evaluation.

MATERIALS AND METHODS Materials

Sodium hyaluronate (HA) was purchased from Lifecore Biomedical (Chaska,Min., 1300 kDa). bFGF was provided by Scios-Nova as a frozen solution(4.3 mg/ml) in 9% sucrose, 20 mM sodium citrate, and 1 mM EDTA adjustedto pH 5. Formulation buffer reagents (sucrose, sodium citrate, EDTA,BSA) were purchased from Sigma. Adipic dihydrazide (AD) and1-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC) were purchased fromAldrich. Sulfo-NHS-Biotin (SNB), 2-(4′ hydroxyphenylazo) benzoic acid(HABA), a 3,3′ diamino benzidine tetrahydrochloride (DAB) metal enhancedsubstrate kit, and an avidin-horseradish peroxidase (Av-HRP) conjugatewere purchased from Pierce. Tween 20 was purchased from Baker.

Biotinylation

The HA-Biotin (HA-Bi) conjugate was prepared by a two step reaction.Hydrazido-HA was synthesized followed by preparation of HA-Bi accordingto the method of Pouyani and Prestwich, Bioconjugate Chem. 5:370-372(1994). Hydrazido-HA was prepared by dissolving 200 mg of HA in 50 ml ofwater. AD (3.5 g) was added to the HA solution and the pH was adjustedto 4.75 with 0.1 N HCl. EDC (382 mg) was added to the solution to beginthe reaction. The pH was monitored periodically and maintained at 4.75by the addition of 0.1 N HCl. The reaction was stopped after 4 hours (atthis point no further increase in pH was detected) by neutralization topH 7 with 1 N NaOH. This product was dialyzed for 72 hrs (Specta/Por,6000 to 8000 MW cutoff) and then lyophilized for 48 hours.

The HA-Bi conjugate was prepared by dissolving 15 mg of Hydrazido-HA in1.5 ml of 0.1 M NaHCO3. The SNB (50 mg) was added to begin the reaction.The solution was stirred with a small magnetic stir bar for 20 hours atroom temperature. The solution was dialyzed for 72 hours and thenlyophilized for 48 hours. The degree of substitution was determined by adisplacement assay according to the manufacturer's protocol (Pierce).Briefly, 900 μL of avidin-HABA reagent was placed in a 1 ml cuvette. Theabsorbance at 500 nm was compared to the absorbance of a solution of 900μL of Avidin-HABA plus 100 μL of a 1 mg/ml HA-Biotin solution. Theaverage degree of substitution was 30 moles of repeating disaccharideunit per mole of biotin.

Formulation

Formulations were prepared by mixing a sterile-filtered solution of bFGFwith solid HA as described in Table 5. The formulation was mixed byrepeated back and forth motion of two syringes connected by a stopcock.Formulations were prepared aseptically and administered in prefilled 1ml plastic syringes with a 21 G needle.

TABLE 5 HA-Bi formulations. Formulations Component HA-Bi + bFGF HA +bFGF HA-Bi basic fibroblast growth  4 mg/ml  4 mg/ml — factor (bFGF)Hyaluronic Acid-Biotin  4 mg/ml —  4 mg/ml Conjugate (HA-Bi) HyaluronicAcid (HA) 16 mg/ml 20 mg/ml 16 mg/ml Sucrose  9%  9%  9% Sodium Citrate20 mM 20 mM 20 mM EDTA  1 mM  1 mM  1 mM

Animal Model Male Sprague-Dawley rats (6-7 weeks old, 160-180 g, n=5 pergroup) were anesthetized with a mixture of acepromazine, xylazine, andketamine. A small incision (5-10 mm) was made laterally in the skin atthe back of the neck. The intersection of the sagittal and lambdoidsutures was located, and 50 μL of each formulation was injected on theleft side with a 21 G needle between the periosteum and parietal.Fourteen days following treatment animals were euthanized by CO₂asphyxiation.

Histology

Tissues for histological evaluation were fixed in 10% neutral bufferedformalin then decalcified in a 13 to 15% solution of EDTA with constant,gentle agitation. Samples were dehydrated and infiltrated with paraffin.Specimens were then embedded in a cross-sectional plane and sectioned at4 μm. Two sections were prepared for each specimen and were stained withhematoxylin and eosin (H&E) or stained for HA with Bi:Av-HRPhistochemistry by the following method. Tissue sections were incubatedfor 30 min in blocker solution (1% BSA/0.05% Tween in PBS) followed by a60 min incubation in detecting conjugate solution (1 μg/ml Avidin-HRP in1% BSA/0.05% tween in PBS). These tissue sections were then placed inwash solution (0.05% tween in PBS) for 5 min. The wash in PBS/tween wasrepeated 5 times with fresh solution. A metal enhanced DAB kit wasutilized to stain for the HA-Bi:Av-HRP complex. Five minutes afterapplying the DAB substrate the sections were rinsed in water. A blackprecipitate formed in the presence of the complex. Finally, thesesections were counterstained with hematoxylin (H) for cellular detail.

Periosteum and Bone Thickness Measurement

The total thickness of the periosteum and parietal bone was determinedat the site of injection. A photograph of each histology section wastaken 2 to 3 mm lateral to the sagittal suture (the approximate midpointbetween the sagittal suture and the edge of the section). Threethickness measurements were taken at the left, middle, and right side ofthe photograph and scaled to determine total bone thickness orperiosteum thickness. Tissue with similar staining characteristics andcell morphology to normal periosteum was included in the periosteumthickness. Both dense cortical bone and new, woven bone were included inthe bone thickness measurement.

Results

Animals treated with bFGF in an HA gel showed an increase in thethickness of the periosteum at 3 days and significant woven boneformation at 10 days. Animals treated without bFGF showed limitedperiosteum thickening and bone formation. HA-Bi was detected in thetissues immediately adjacent to the thickened periosteum at 3 days andthe newly formed bone at 10 days.

For HA-Bi, Administration at 3 Days

At the injection site a distinct mass of HA was present above theperiosteum. There was a portion of the periosteum elevated from thelamellar bone from the surgical trauma. In the area stained for HA,there was a localized area of fibrous tissue and a non-specific cellularinfiltrate in which lymphocytes and degenerating cells were evident. Thesurrounding tissue consisted of fine fibrous tissue.

HA-Bi, Administration at 10 Days

The HA-Bi treated animals showed normal lamellar bone with an area ofnon-specific fibrous tissue resembling granulation tissue above it. Thisarea contained lymphocytes, fine blood vessels, fat cells and a fewfragments of unstained material. The brownish-black peroxidase stain waswithin the dense fibrous tissue superficial to the calvarium on the leftside. The HA was distributed non-specifically within the fibrous tissue.

For HA-Bi+bFGF Administration at 3 Days

At the injection site there was hyperplasia of the periosteal layeroverlaying the pre-existing lamellar bone. Quantitatively, theperiosteum in HA-Bi+FGF treated animals was 403% greater than animalstreated only with HA-Bi gel. A mass of vascularized, exuberant fibroustissue was present above the thickened periosteum. Within this fibroustissue, fat cells were present and a non-specific inflammatory cellinfiltrate containing some polymorphonuclear leukocytes, histocytes andplasma cells were present. The residual HA extended across the midlinesuture and appeared to be undergoing encapsulation. These samples showeda concentration of brownish-black stained material (i.e. HA) mainlyconcentrated within the confines of the encapsulated tissue. More ofthis material appeared to be non-specifically retained within a fibrousnetwork and some appeared to be non-specifically accumulated within thecytoplasm of local histocytes.

HA-Bi+bFGF, Administration at 10 Days

The injection site showed that the preexisting calvarial lamellar bonewas covered by a thick layer of maturing woven bone which was normal instructure and staining qualities. The total bone thickness was 70%greater in animals treated with HA-Bi+FGF than in animals receivingHA-Bi gel. This new bone typically extended just beyond the midlinesuture onto the right side of the calvarium. DAB staining for HA wasseen in the superficial layers of the fibrous tissue proliferationsurrounding the newly formed woven bone. The peroxidase stainingindicated that the HA was typically present in tissues adjacent to newlyformed bone. Above the woven bone a fibro-periosteal layer was present.Superficial to this there was an extensive area of fine fibrous tissuewhich was vascularized and contained adipose cells. Some lymphocytes,plasma cells, and histocytes were also present in this well developedarea which was limited by a thin fibrous tissue layer.

HA+bFGF Administration at 3 Days and 10 Days

Qualitatively and quantitatively conjugating biotin to HA had no effecton the biological response to the formulation (Table 6). The periosteumand bone thicknesses of the HA+FGF treated group were not statisticallydifferent from the HA-Bi+FGF treated group (p>0.05), but weresignificantly different than HA-BI treated controls (p<0.001).Histologically, the animals treated with HA+bFGF with no biotin weresimilar to the HA-Bi+bFGF except that there were no areas that werestained brownish-black from the DAB substrate. These areas were notexpected to stain because of the absence of biotin. A few cells didstain positively because of the presence of endogenous peroxidaseactivity.

TABLE 6 The periosteum and bone thickness of the three groups examinedin this study Periosteum thickness, Total bone thickness, 3 days (μm) 10days (μm) % greater % greater than than Treatment mean ± SD HA-Bi gelmean ± SD HA-Bi gel HA-Bi + bFGF 53 ≅ 3 403% 464 ± 21 70% HA + bFGF 47 ±2 341% 420 ± 16 54% HA-Bi gel 11 ± 1  0% 272 ± 6   0%

The administration of an HA+bFGF gel by subperiosteal injection had asignificant effect on the proliferation of the periosteum and activebone formation. Three days after administration, the periosteum wasnearly 5 fold thicker than control. In addition, 10 days followingadministration the parietal bone thickness was 70% greater than controlin HA/bFGF treated rats. The HA carrier in the formulations examinedhere directs the formation of new bone by placement of the material; HAis seen in areas of active bone formation. Following injection ofHA+bFGF, the HA provides a reservoir of bFGF adjacent to the site of newbone formation.

In addition to providing site directed release of bFGF, HA hasbiological properties that appear to support an environment to promotebone formation. HA may have a synergistic effect with FGF.

The foregoing disclosure and description of the invention areillustrative and explanatory thereof and various changes in the size,shape and materials as well as in the details of the preferredembodiment may be made without departing from the spirit of theinvention.

What is claimed is:
 1. A composition for treatment of diseased, injuredor abnormal bone composition comprising an effective amount of a mixtureof a growth factor and hyaluronic acid sufficient to enhance bone growthrate and magnitude and having sufficient viscosity and biodegradabilityto persist upon application at an intra-articular site of desired bonegrowth for a period of time sufficient to enhance said bone growth rateand magnitude.
 2. A composition according to claim 1 wherein saidhyaluronic acid is uncrosslinked.
 3. A composition according to claim 1wherein said composition comprises 0.1 to 4% by weight of hyaluronicacid in solution.
 4. A composition according to claim 1 wherein saidgrowth factor comprises bFGF.
 5. A composition according to claim 4wherein said bFGF is present in said composition in a range of about10⁻⁶ to 100 mg/ml of said composition.